Metagenomic analysis of viruses in reclaimed water
Increase in urbanization and population growth puts pressure on the limited water resources available. This has led many states all over the world to look for alternative sources of water. Reclaimed water or reclaimed water, which is the re-usable end product of waste water treatment, has been used as an alternative water source in the recent years (1). This waster is not usually used for direct human consumption but is used for purposes like irrigation, recreation, recharge ground water etc. The major concern with reclaimed water is safety issues as the microorganisms in the water have not been completely characterized. A group of scientists in Florida set to do a metagenomic analysis of the reclaimed water used in Florida (1). Since viruses are highly stable, and quite a lot can survive the harsh conditions of waste water treatment, this group set to a metagenomic analysis of viruses in the reclaimed water. Methodology There were four samples of water analyzed in this study: *potable water for comparison. *reclaimed water from the waste water treatment facility at its point of discharge (Effluent). *reclaimed water from a public park sprinkler (Park). *reclaimed water from a plant nursery (Nursery). All the water samples were processed within three hours of collection. Enumeration and Visualization of virus like particles (VLPs) Samples were collect in 50 ml tubes, fixed with 2% paraformaldehyde and then passed through 0.02µm Anodisc filters to collect the VLPs. The filters were then stained with SYBR Gold and counted to get the numbers of VLPs. To visualize the VLPs, transmission electron microscopy was done (Figure 1B). Nucleic acid isolation, library construction, sequencing and Bioinformatics The water samples collected were concentrated and purified using a combination of different methods like tangential flow filtration, density-dependent centrifugation and nuclease treatment followed by cesium chloride centrifugation. A 0.2µm filter to get rid of large particles, eukaryotes, bacteria and other microorganisms. Nucleic acids were then isolated using a kit, followed by separation of DNA and RNA. RNA samples were treated with DNase I. For the RNA samples, cDNA were made. DNA and cDNA were pyrosequenced and libraries made. Sequences larger than 100nt were used to construct contigs using SeqMan (DNASTAR, Madison, WI). Sequences smaller than 200nt were not used to do BLASTX search against the GenBank non-redundant (nr) protein database, with an E value <0.001 considered significant match. These BLASTX results were analysed using the Metagenome Analyzer (MEGAN) software (2). Contigs that showed a match to cellular organisms were further analyzed or sequence similarity using the ACLAME (‘A Classification of Mobile Genetic Elements’) database. Results The morphology of various kinds of VLPs found in reclaimed water using transmission electron microscopy is shown in Figure 1B. Quantification of VLPs from different water sources showed approximately 1000-fold higher number of VLPs in reclaimed water when compared to potable water (Figure 1A hits.gif|Figure 3: Analysis of contigs greater than 200nt in length using BLASTX against GenBank and ACLAME databases. phages.gif|Figure 4: Distribution of metagenomic sequences of potable versus reclaimed water among phage families. Source: reference (1). ). To figure out the type of VLPs in reclaimed water compared to potable water, sequence analysis on these VLPs were done. BLASTX search for these sequences show that 70% of the DNA VLPs and 57% of the RNA VLPs (compared to 44% of Potable water DNA VLPs) have no hits to the GenBank sequence database, identifying them as novel VLPs (Figure 3A). Further analysis was done on the contigs that had a match to the GenBank database (assigned contigs) and these were compared against the ACLAME database too. These sequences were assigned to biological groups based on the sequence similarity of the viral sequences to the sequences to the database. A lot of the sequences match protein sequences from bacteria and plasmids, i.e., matched to mobile genetic elements (Figure 3B). The purification methods of cesium chloride centrifugation should have got rid of residual plasmids that would have survived other treatments. Therefore, these don’t seem to be contaminating sequences. A possible explanation is that a lot of these could be phage sequences due to their similarity to plasmid like sequences and other host sequences as a result of lateral transfer (3, 4). Also, bacteriophages acquire sequences from the bacterial host, which explain the presenc of bacteria like proteins. Other viruses could be novel having plasmid like sequences. These sequences were then analyzed to determine their host. Potable and reclaimed water DNA VLPs had similar results with most hosts being prokaryotes. The reclaimed RNA sequences had a diverse host range (Figure 3C). Notable, no human pathogens were identified in any of the metagenomic analysis of reclaimed water. Bacteriophages seem to dominate the DNA VLPs based on the sequence data. These were looked at in more detail to analyze individual bacteriophage families for the reclaimed and potable DNA VLP sequences. The reclaimed and potable VLPs seemed to have a different individual host distribution within the same phage family (Figure 4). Conclusion No human pathogenic viral sequences were found in this study of reclaimed water using metagenomin analysis. Further characterization of reclaimed and potable water viral metagenomes needs to be done as there were several novel viral sequences identified. References 1. Rosario, K., et al., Metagenomic analysis of viruses in reclaimed water. Environ Microbiol, 2009. 11(11): p. 2806-20. http://www.ncbi.nlm.nih.gov/pubmed/19555373 PubMed 2. Huson, D.H., et al., MEGAN analysis of metagenomic data. Genome Res, 2007. 17(3): p. 377-86. http://www.ncbi.nlm.nih.gov/pubmed/17255551 PubMed 3. Hazen, T.H., et al., Sequence characterization and comparative analysis of three plasmids isolated from environmental Vibrio spp. Appl Environ Microbiol, 2007. 73(23): p. 7703-10. http://www.ncbi.nlm.nih.gov/pubmed/17921277 PubMed 4. Osborn, A.M. and D. Boltner, When phage, plasmids, and transposons collide: genomic islands, and conjugative- and mobilizable-transposons as a mosaic continuum. Plasmid, 2002. 48(3): p. 202-12. http://www.ncbi.nlm.nih.gov/pubmed/12460536 PubMed